Human cytomegalovirus (HCMV) infection permanently afflicts over half of all Americans, is the most common infectious cause of birth defects, and threatens the lives of thousands of children and adults with impaired immune systems. New strategies to stop the infection are urgently needed because current antiviral drugs are inadequate and a vaccine is non-existent. Our ultimate goal is to discover ways of immediately blocking HCMV infection, which may be reasonably achieved through selective disruption of viral gene expression. Toward this goal, the objective of this research project is to determine the mechanisms utilized by HCMV to regulate transcription of its genome during the course of an active infection. We hypothesize that HCMV adopts the basic host principles of transcription control of the RNA polymerase II, but modifies this host machinery through the actions of viral products. We further hypothesize that the major HCMV MIE proteins are central to orchestrating the temporal cascade of viral transcription, while also differentially modulating host transcription. The planned experiments will examine in-depth the transcriptional regulation of both HCMV and host genomes using powerful global techniques (ChIP-Seq, PRO-Seq, and RNA-Seq), novel in vitro systems (customized nuclear walk-on assays), originally crafted reagents (HCMV mutants), and a newly developed technology for rapidly and robustly eliminating proteins of interest to unravel their role in HCMV transcriptional regulation. The plan integrates the biochemical and molecular expertise of the Price Lab in transcription control and global techniques and the extensive experience of Meier Lab in HCMV virology and gene expression. The results of these experiments will be the first to determine the extent to which HCMV applies recently discovered mechanisms controlling animal gene expression, as well as the roles of the HCMV MIE proteins in commandeering this vital process. Results obtained through the combined efforts of the Meier and Price Labs will be applicable to understanding transcriptional regulation not only of HCMV, but all DNA viruses. The work proposed here will set the groundwork for future studies aimed at controlling infections of HCMV, HSV, EBV, and other viruses.